Echo contrast agent

ABSTRACT

An aqueous preparation for receiving and stabilising micro gas bubbles for use as echo contrast media containing polyoxyethylene/polyoxypropylene polymers and negatively charged phospholipids, which is suitable for demonstration of the left ventricle, is indicated.

This application is a continuation of application Ser. No. 08/078,189,filed Jun. 21, 1993, now abandoned, which is a 371 of PCT/EP92/00012,filed Jan. 4, 1992.

FIELD OF THE INVENTION

The invention relates to an aqueous preparation for receiving andstabilising micro gas bubbles for use as echo contrast media.

PRIOR ART

Since ultrasound is highly reflected by gas bubbles suspended inliquids, there was an early proposal to use aqueous preparations whichcontain stabilised micro gas bubbles as contrast media for ultrasonicdiagnosis. Micro gas bubbles can be stabilised in aqueous preparationsby reducing the surface tension, that is to say by adding suitablesurfactants.

It is indicated in EP-B-0 077 752 that aqueous solutions which contain asurfactant or a mixture of surfactants and, in addition, aviscosity-increasing substance have advantageous contrast-generatingproperties. Indicated as suitable surfactants are, inter alia, non-ioniclecithins and lecithin fractions, and polyoxyethylene/polyoxpropylenepolymers. The preparations indicated in the six preparation examples ofEP-B-0 077 752 each contain as surfactant apolyoxyethylene/polyoxypropylene polymer and as viscosity-increasingsubstance glucose or dextran or the polyoxyethylene/polyoxypropylenepolymer itself. Repetition of the preparation examples has shown thatthe contrast-generating action is unsatisfactory. Thus, the preparationsof EP-B-0 077 752 are unsuitable for demonstrations of the leftventricle.

It has now been found, surprisingly, that aqueous preparations which,besides polyoxyethylene/polyoxypropylene polymers, contain negativelycharged phospholipids are outstandingly suitable for receiving andstabilising micro gas bubbles.

SUMMARY OF THE INVENTION

The invention therefore relates to aqueous preparations for receivingand stabilising micro gas bubbles for use as echo contrast mediacontaining polyoxyethylene/polyoxypropylene polymers and negativelycharged phospholipids.

Further subject-matter is evident from the claims.

Preferred polyoxyethylene/polyoxypropylene polymers are those with anaverage molecular weight of 8,350 to 14,000.Polyoxyethylene/polyoxypropylene polymers are also called poloxamers andare commercially available, for example, under the proprietary namePluronics® (Wyandotte Chemicals Corp.). The preparations according tothe invention contain 0.1 to 10%, preferably 1 to 5%, ofpolyoxyethylene/polyoxypropylene polymers. The content of negativelycharged phospholipids is from 0.01 to 5%, preferably 0.5 to 2%.Percentage data in each case relate to weight/volume.

Suitable negatively charged phospholipids are phosphatidylglycerols,phosphatidylinositols, phosphatidylethanolamines and phosphatidylserinesand the lyso forms thereof. By lyso forms of the negatively chargedphospholipids are meant negatively charged phospholipids which containonly one acyl radical. Lyso forms of the negatively chargedphospholipids in which the acyl group is bonded to the oxygen of thecarbon atom 1 of the glycerol molecule are preferred. Particularlypreferred negatively charged phospholipids aredipalmitoylphosphatidylglycerol (DPPG) anddistearoylphosphatidylglycerol (DSPG), anddistearoylphosphatidylglycerol (DSPG) is very particularly preferred.

The preparations according to the invention are distinguished from thoseof the prior art in that echo contrast media containing micro gasbubbles can be produced with little mechanical effort and, because oftheir great stability, generate a long-lasting contrast and moreover areoutstandingly suitable for demonstration of the left ventricle. Itshould be particularly emphasised that the preparations according to theinvention are excellently suitable for the demonstration of internalsurface structures because the micro gas bubbles apparently adhere wellto surfaces and thus generate informative contrast even after the microgas bubbles which are located in the lumen of vessels have been flushedout. This makes it possible, for example, to demonstrate more clearlythe dynamics of the heart even after the contrast medium has been washedout.

The preparation of the preparations according to the invention is notdifficult and can take place by introducing the individual componentstogether or successively into water and dissolving, if necessary withheating and stirring. Sterilisation is also possible if required, forexample by heat sterilisation.

Glycerol, mannitol and ammonium salts of amino acids, preferablyglycine, have proved particularly well suited for adjusting theisotonicity of the preparations according to the invention.

The micro gas bubbles are generated in a manner known per se andexpediently only shortly before administration to the patients to beinvestigated. If, for example, the preparation according to theinvention is provided in a vial, the solution can be drawn up togetherwith the required amount of air into a conventional syringe and injectedagain into the vial through a narrow needle with the highest possiblepressure. If necessary, the drawing up and expulsion from the syringe isrepeated several times. It is also possible as an alternative to forcethe preparations according to the invention backwards and forwardsbetween two syringes via a connector with a narrow cross-section or amixing chamber inserted between the two syringes. The latter methodleads to particularly informative ultrasonic images with, at the sametime, a further increase in productivity.

Suitable gases for generating the micro gas bubbles are allphysiologically tolerated gases. The preparations according to theinvention are converted into a foam with 0.01 to 0.1, preferably with0.04 to 0.06, ml of gas per 1 ml. They are preferably administeredintravenously after generation of the micro gas bubbles. Depending onthe purpose for which the preparations according to the invention areused, 1 to 20 ml, preferably 2 to 8 ml, and particularly preferably 5 mlare administered.

It should be particularly emphasised that lower doses of thepreparations according to the invention are possible because of theirincreased productivity compared with the prior art.

EXAMPLES

1. 3.0 g of polyoxyethylene/polyoxypropylene polymer with an averagemolecular weight of 8,400 (Pluronic®F68), 1.0 g ofdipalmitoylphosphatidylglycerol (DPPG) and 3.6 g of glycerol areintroduced into 80 ml of water. The mixture is heated to about 80° C.and stirred until complete dissolution has taken place. After cooling,the volume is made up to 100 ml with distilled water.

2. The process is carried out as in Example 1 with the difference that1.0 g of soya phosphatidylglycerol (supplied by Lucas Meyer, Hamburg) isused in place of DPPG.

3. 1.1 g of glycine are introduced into 80 ml of water. A pH of 6 to 7is adjusted with dilute ammonia. 3.0 g ofpolyoxyethylene/polyoxypropylene polymer with an average molecularweight of 8,400 (Pluronic®F68) and 1.0 g of DPPG are added to thesolution. The mixture is heated to about 80° C. and stirred untilcomplete dissolution has taken place. After cooling, the volume is madeup to 100 ml with distilled water.

4. The process is carried out as in Example 3 with the difference that1.0 g of soya phosphatidylglycerol (supplied by Lucas Meyer) is used inplace of DPPG.

5. 4.0 g of polyoxyethylene/polyoxypropylene polymer (Poloxamer 188,Pluronic®F68), 1.0 g of distearoylphosphatidylglcyerol and 5.4 g ofmannitol are introduced into 80 ml of water. The mixture is heated toabout 80° C. and stirred until dissolution is complete. After cooling,the volume is made up to 100 ml with distilled water.

COMPARATIVE EXPERIMENTS

The investigations were carried out on conscious male beagle dogs(18.2-30.5 kg body weight). The dogs received in each case 5 ml,administered i.v., of the contrast medium preparations described below:

A: An infusion solution, containing 35 g of crosslinked polypeptides per1,000 ml, for plasma replacement (Haemaccel® supplied by Behringwerke)

B: Echovist® (echo contrast medium supplied by Schering)

C: An aqueous solution containing 4% by weight poloxomer 188(Pluronic®F68) and 4% by weight glucose (Example 1 in EP 0 077 752)

D: An aqueous solution containing 2% by weight poloxamer and 4% byweight glucose (Example 2 in EP 0 077 752)

E: An aqueous solution containing 1% by weight poloxamer and 4% byweight glucose (Example 3 in EP 0 077 752)

F: Preparation according to the invention from Example 5

Solutions A, C, D, E and F are drawn up without air into a firstsyringe. This syringe is then connected by the free end to a mixingchamber which contains 0.18 ml of air and is firmly connected to asecond syringe. Immediately before administration, the solutions arepumped out of the first syringe through the mixing chamber into thesecond syringe and back again five times.

The commercially available contrast medium B is prepared as instructedin the pack insert.

The echocardiographic ultrasonic scans were carried out with a Sonoscope4 ultrasonic instrument with mechanical head at 3.5 MHz. The videoprintsof the resulting ultrasonic images were evaluated for the intensity ofcontrast by densitometry. The densitometer used (Gretag D182) determinesthe changes in the brightness in 100 steps in the range from 0.00 to2.50 density units. The calibration is carried out using the DIN 16536calibration card (calibration reference) provided by the manufacturer,where the lightest white is assigned the value 1.64 and the darkestblack is assigned the value 0.00. The average of four individualdeterminations on an area of 1 cm×1 cm gives the value for theadministered preparation for each animal.

The results obtained are shown in the table which follows.

    ______________________________________                                        right ventricle    left ventricle                                                       Intensity         Intensity                                         5 ml   Contrast max    10 sec                                                                              Contrast                                                                             max  10 sec                               ______________________________________                                        A      yes      1.18   0.86  no     0.00 0.00                                 B      yes      1.09   0.65  no     0.00 0.00                                 C      yes      1.20   0.78  no     0.00 0.00                                 D      yes      1.23   0.87  no     0.00 0.00                                 E      yes      1.22   0.93  no     0.00 0.00                                 F      yes      1.19   0.82  yes    0.78 0.72                                 ______________________________________                                    

Intensity in Density Units (DU)

It is evident from the results that the echo contrast media according tothe invention enter the lungs, in contrast to the echo contrast media ofthe prior art, and are therefore excellently suited for diagnosis in theleft ventricle. The utilisability of ultrasonic imaging in cardiacdiagnosis is considerably extended by the echo contrast media accordingto the invention.

In addition, it has been found that the micro bubbles of the echocontrast media according to the invention apparently have a considerableaffinity for the internal surfaces of vessels and cavities in the body.The consequence of this is that the outlines of vessels and cavities aredemonstrated much better and therefore more informatively than waspossible with contrast media of the prior art. It is particularlyadvantageous in this connection that this great improvement in thedemonstration of the surfaces of vessels and cavities even persists whenthe lumen of the vessel or cavity is already free of echo contrastmedium. This surprising contrast of surfaces can be utilised, forexample, for observation of the endocardium.

FIGS. 1 and 2 depict the result of an experiment to demonstrate thisnovel contrast of surface structures.

FIG. 1 shows the echocardiographic image of the endocardium of aconscious beagle dog in the so-called four-chamber view immediatelybefore appearance of the first contrast after administration of 1 ml ofecho contrast medium from Example 1.

FIG. 2 shows the endocardium of the animal after the echo contrastmedium has already been washed out of the heart again.

It is evident from comparison of the two figures that an unexpectedmarking of the endocardium, which signifies a large gain in informationfor diagnostic purposes, is possible with echo contrast media accordingto the invention.

We claim:
 1. An aqueous preparation useful for receiving and stabilisingmicro gas bubbles for use as echo contrast media and containingpolyoxyethylene/polyoxypropylene polymer and anionic phospholipid.
 2. Anaqueous preparation of claim 1 which comprises, as sole essentialcomponents, water, polyoxyethylene/polyoxypropylene polymer and anionicphospholipid, optionally in combination with isotonicity adjustingcomponent means.
 3. A preparation according to claim 2, wherein the thepolyoxyethylene/polyoxypropylene polymer comprises from 0.1 to 10%(weight/volume).
 4. A preparation according to claim 3, wherein thepolyoxyethylene/polyoxypropylene polymer comprises from 1 to 5%(weight/volume).
 5. A preparation according to claim 1, containingphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine orphosphatidylserine as the anionic phospholipid.
 6. A preparationaccording to claim 5, containing distearoylphosphatidylglycerol as theanionic phospholipid.
 7. A preparation according to claim 1, containingthe anionic phospholipid in an amount of from 0.01 to 5%(weight/volume).
 8. A preparation according to claim 1, containing 3%(weight/volume) of polyoxyethylene/polyoxypropylene polymer with anaverage molecular weight of 8,400 and 1% (weight/volume) ofdistearoylphosphatidylglycerol.
 9. A preparation according to claim 1,wherein the anionic phospholipid is present as lyso form.
 10. A processfor the preparation of an aqueous preparation for receiving andstabilising micro bubbles for use as echo contrast media, whichcomprises dissolving in water polyoxyethylene/polyoxypropylene polymertogether with a anionic phospholipid and customary auxiliaries forachieving isotonicity.
 11. In an aqueous echo contrast medium containingpolyoxyethylene/polyoxypropylene polymer, the improvement wherein thepolymer is in admixture with anionic phospholipid.
 12. An aqueouspreparation of claim 2 which consists essentially ofpolyoxyethylene/polyoxypropylene polymer and anionic phospholipiddissolved in water.
 13. An aqueous preparation of claim 2 useful forreceiving and stabilizing micro gas bubbles for use as echo contrastmedium and containing polyoxyethylene/polyoxypropylene polymer andanionic phospholipid;the polymer having an average molecular weight offrom 8,350 to 14,000; and the anionic phospholipid being a memberselected from the group consisting of a phosphatidylglycerol, aphosphatidylinositol, a phosphatidylethanolamine and aphosphatidylserine.
 14. An aqueous preparation according to claim 13containing from 0.1 to 10 percent (weight/volume) of thepolyoxyethylene/polyoxypropylene polymer.
 15. An aqueous preparationaccording to claim 14 containing from 0.01 to 5 percent (weight/volume)of the anionic phospholipid.
 16. An aqueous preparation according toclaim 15 wherein the anionic phospholipid is a phosphatidylglycerol. 17.An aqueous preparation according to claim 15 wherein the anionicphospholipid is a phosphatidylinositol.
 18. An aqueous preparationaccording to claim 15 wherein the anionic phospholipid is aphosphatidylethanolamine.
 19. An aqueous preparation according to claim15 wherein the anionic phospholipid is a phosphatidylserine.
 20. Anaqueous preparation of claim 1 which is suitable for demonstration ofthe left ventricle.
 21. A preparation according to claim 1, wherein thepolyoxyethylene/polyoxypropylene polymer has an average molecular weightof from 8,350 to 14,000.
 22. An echo contrast medium which is an aqueouspreparation of claim 1 in intimate admixture with stabilized micro gasbubbles.
 23. An echo contrast medium which is an aqueous preparation ofclaim 21 in intimate admixture with stabilized micro gas bubbles.
 24. Anecho contrast medium of claim 22, having great stability and beingproduced with little mechanical effort.